Epigenetically Reprogramming of Human Embryonic Stem Cells by 3-Deazaneplanocin A and Sodium Butyrate

Objectives: Infertility affects about 6.1 million women aged 15-44 in the United States. The leading cause of infertility in women is quantitative and qualitative defects in human germ-cell develop-ment (these sentences are not mentioned in introduction so it is not correct to mention in abstract, you can omit). Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of developing blastocysts and have a broad clinical potential. hESCs have been classified into three classes based on their epigenetic state. The goal of this study was to epigenetically reprogram Class II and Class III cell lines to Class I (naïve state), and to in vitro differentiation of potent hESCs to primordial germ cells (PGCs). Methods: Recent evidence suggests that 3-deazaneplanocin A (DZNep) is a global histone methylation inhibitor which selectively inhibits trimethylation of lysine 27 on histone H3K27, and it is an epigenetic therapeutic for cancer. The characteristics of DZNep lead us to hypothesize that it is a good candidate to epigenetically reprogram hESCs to the Class I. Additionally, we used sodium butyrate (NaBu) shown in previous studies to up-regulate the expression of germ cell specific markers (these sentences should be come in introduction). Results: We used these two drugs to produce epigenetically stable hESC lines. hESC lines are an appropriate system for disease mod-eling and understanding developmental stages, therefore producing stable stem cell lines may have an outstanding impact in different research fields such as preventive medicine. Conclusions: X-Chromosome inactivation has been used as a tool to follow the reprogramming process. We have used immunostaining and western blot as methods to follow this reprogramming qualitatively and quantitatively.


INTRODUCTION
Human embryonic stem cells (hESCs) can be maintained in culture in a self-renewing state and differentiate into all three embryonic germ layers. Although they hold great promise for regenerative medicine, hESC-based therapy fac-es several challenges. Concerns have been raised regarding their genetic stability. Although not fully understood, challenges also exist over epigenetic stability. Epigenetic changes make nuclear program altered without changing the primary DNA sequence. Because epigenetic www.mui.ac.ir changes can substantially modify cellular behavior and are mitotically and meiotically heritable, investigation of the epigenetic properties of human hESC is desirable prior to considering their use in vivo. Epigenetic state is one of the hESCs states that enables stem cells with the unique properties to self renew or differentiate into any cell type in the body. The hESC state may be influenced by the manner in which ESCs are derived and maintained. Recent studies have showed that the efficiency of induced pluripotent stem (iPS) cells formation is enhanced upon addition of valporic acid, an inhibitor of histone deacetylases, to the culture medium. Sodium butyrate, a naturally occurring short-chain fatty acid, supports the extensive self-renewal of mouse embryonic stem cell (hESCs) and . 1 X-chromosome inactivation (XCI) phenomenon has been used to examine the epigenetic stability of hESC. Because XCI is one of the first measurable epigenetic changes in the early mammalian embryo and is coincident with differentiation, XCI marker serves as an excellent tool to investigate the epigenetic behavior of hESC.XCI is a mechanism to compensate gene load difference between XY males and XX females in mammals. During early embryogenesis, one of two X-chromosomes in every cell is inactivated, and stably inherited through cell division of somatic cells. 2 XIST makes a noncoding RNA required to initiate silencing during XCI. Before XCI in mESCs, XIST is expressed at low level. Upon cell differentiation and the onset of XCI, XIST RNA is transcriptionally induces and forms a cloud around the inactive X (Xi) .However, it is known that the two X chromosomes are active in oocytes, indicating that the inactive X chromosome must be reactivated during germ cell development. The reactivation of inactive X chromosome occurs at least twice during mammalian development, once in the epiblast cell lineage at the peri implantation stage and once in the primordial germ cells (PGCs) at the midgestation. 2 The dynamics of X chromosome activity is tightly correlated with major genomic reprogramming events occurring during mammalian development.
Recently, 11 distinct hESC lines have been studied in order to investigate their epigenetic properties by using XCI markers mainly studying XIST expression.Unlike mESCs, hESCs are pre-XCI and there is variability of XIST expression among different hESC lines. 3 These cell lines can be subgrouped into three classes. Class I line has the capacity to recapitulate XCI when induced to differentiate in culture. Class II cells have already undergone XCI. In class III cell lines, despite losing tirmethylation of histone H3-K27 (H3K27me3), there is a tendency to lose XIST RNA expression.
Histone lysine methylation has been shown to index silenced chromatin regions at pericentric heterochromatin or of the inactive X chromosome. 4 H3K27me3 is a repressive chromatin mark. Recently, 3-deazaneplanocin A (DZNep) was discovered to selectively inhibit H3K27me3. DZNep affects multiple histone methyltransferases and can epigenetically reactivate a different cohort of genes. 5 In the current study, our goal was to reprogram class II cell lines to class I by using DZNep as H3K27me3 methyltransferase inhibitor, and sodium butyrate as histone deacetylase inhibitor which enhances self-renewal status of embryonic stem cells and also up-regulates germ-cell specific markers. 1 We used three Class II hESC lines: HSF6-8, HSF6-10, and HSF6-S9.

Histone Protein Extraction
Human Embryonic stem cells were harvested and washed twice with ice-cold PBS. Cells were re-suspended in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X 100 (v/v), 2mM phenylmethylsufonyl fluoride (PMSF), 0.02% (w/v) NaN2) with a cell density of 10 7 cells per ml. Cells were lysed for 10 min using gentle stirring, and centrifuged at 6500x g for 10 minutes at 4 degree C to spin down the nuclei. Pellet was re-suspended in 0.2 N HCl at a density of 4x10 7 nuclei per ml. The extract was stored overnight at 4 degree C. Samples were centrifuged at 6500xg for 10 minutes to pellet debris. Supernatant was stored and the histone concentration was detected by Commassie Blue.

Western Blots
Cells were harvested by treatment with Try-pLE express. Cells were lysed using 1.0 ml hywww.mui.ac.ir potonic lysis buffer (10mM Tris-HCl, 1.5mM MgCl 2 10mM KCl, 0.34M sucrose, 10% glycerol,40µl of PI stock solution, and 1.0 µl of DTT). Nuclear histones were separated on 16% SDS-polyacrylamide gels at 200v, 150mA for an hour and stained with commasie blue. Equal amounts of protein were separated in SDSpolyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were probed with antibodies against histone H3, H3K27me3, and H3K4me3.

Differential Expression of XCI Marker in Cultures of Female hESC Lines
Using immunostaining of H3K27me3, cultures of female HSF6-8, HSF6-10, and HSF6-S9 cells showed 0-50% of XCI marker. Cells were stained with DAPI (a dye binding to DNA), Oct4 (a stem cell marker), and H3K27me3. In a subset of stem cell population, H3K27me3 accompanied Xi, however, some H3K27me3 didn't accompany Xi (Fig.1). The absence of H3K27me3 marker is not indicative of X chromosome loss. X chromosome DNA FISH in a subset of cells which didn't have H3K27me3 enrichment along with Xi, showed the presence of two X chromosomes (Fig. 2).

Optimization of DZNep Concentration and Colony Morphology
In cancer treatment, DZNep is used with high concentration (10µM) in order to induce apoptosis in cancerous cells, and reactivate genes which have became silent by cancer development. Therefore, in the current study the DZNep concentration should have been optimized. NaBu was added at 0.2 mM during three passages and then different concentrations of DZNep were added. The optimal concentration for growing colonies of DZNep seemed to be 0.1 µM (Fig. 3). Healthy, undifferentiated HSF6-8, HSF6-10, and HSF6-S9 colonies treated with 0.2µM NaBu, and 0.1µΜ DZNep seemed to have well-defined uniform borders and the individual cells within the colony appeared to be similar. More cells per colony were observed compared to the untreated colonies (Fig. 4).

hESCs Treated With DZNep Show Tendency to Lose XCI H3K27me3 Marker
Immunostaining analyses showed that cells tend to lose H3K27me3 enrichment associated with Xi by several passages. This effect was prominent when NaBu was added to cells for 11 passages. Cells lost H3K27me3 marker by 70%. Treating cells with DZNep for five passages caused cells to lose H3K27me3 by 90% (Fig. 5).  www.mui.ac.ir    Figure 6. X-Chromosome is associated with Euchromatic region in reprogrammed cells. A: DNA FISH using an X chromosome specific probe was performed on HSF1 male cell line. All cells have one active X chromosome. H3K4me3 (a euchromatic region marker) is shown in red. B: H3K4me3 immunostaining and DNA FISH for X chromosome in HSF6 (10) treated cells show two active X chromosomes. C: Some portion of HSF6 (10) treated cells shows resistance to reprogramming and still has one inactive X-chromosome.

X-Chromosomes are associated with Euchromatic Region in Reprogrammed Cells
HSF1 male cell line immunostained with H3K4me3 (a euchromatic region marker) showed homogenous red color with no exclusion mark which is indicative of a heterochromatic region. X-chromosome DNA FISH showed one active X chromosome (Fig. 6A). In a subpopulation of HSF6-10 cell line, there was a homogenous staining for H3K4me3 with no exclusion mark, and DNA FISH showed the presence of two active X chromosomes (Fig.  6B). In another subset of HSF6 (10) cell line, there was an exclusion mark in H3K4me3 stain-ing, and DNA FISH showed two X chromosomes (Fig. 6C).

Western Blot Analyses Showed no Global Changes in Trimethylation of H3K27 and H3K4
Control cells had no drug added. NaBu and DZNep were added to treated cells with O.2µΜ and 0.1µM concentrations respectively. Western analyses of HSF6-8, HSF6-10, and HSF6-S9 control cells, treated cells with NaBu, and treated cells with NaBu and DZNep showed no global changes in trimethylation of H3K27 and H3K4. Histone H3 was the loading control. The trimethylation of H3K27 demonstrated no obvious changes in control and treated cells (Fig. 7)  www.mui.ac.ir

DISCUSSION
In order to mimic in vivo germ cell development pathway, hESCs should be in their native state, and keep their native state during passages. Three classes of hESC lines derived from ICM of developing blastocyst are similar in their pluripotency potential and forming teratomas when injected to mice. The different point among them is H3K27me3 enrichment deposited in Xi. Also, these three classes have different expression of XIST RNA. Class I cell lines with active X chromosomes are the ones which resembles the in vivo system. However, hESC lines are usually a kind of class II and class III, and even if they are class I, they would transform to class II or III through passaging.
Treated cells with 0.2mM NaBu and 0.1mM DZNep formed colonies with typical stem cell colony appearance which indicates that these two drugs would not interfere with self-renewal state of the cells. Additionally, treated cells had more cells per colony which suggests that the drugs even promote the self-renewal state. Less differentiation in treated cells compared the controls suggests that the differentiation pathway would stop or slow down by NaBu and DZNep.
The loss of H3K27me3 associated with Xi confirmed by immunostaining analyses suggests that NaBu and DZNep have ability to change H3K27 modification. H3K4me3 staining confirmed the existence of two active X chromosomes. In H3K4me3 staining there was no exclusion mark which suggests that both X chromosomes were active. Immunostaining results indicate that 30% of treated class II cells have been reprogrammed to Class I.
H3K27me3 is a suppressing marker. Naturally, many genes should be kept silent, such as oncogenes. Paternally or maternally expressed genes should become silent in the other parent. Therefore, the integrity maintences of necessary silent genes are crucial. Western blot analyses showed that NaBu and DZNep didn't change the global trimethylation of H3K27 and H3K4, and they selectively change the trimethylation of H3K27 associated with Xi. Therefore, using these two drugs with mentioned concentrations is safe, and won't have any global effect.
For future direction, we want to perform differentiation assays to confirm the proper reprogramming process. Also, gene expression assays seem to be informative for investigating gene expression profiles between control and treated cells. In order to rule out any chromosomal abnormality causing by DZNep and NaBu, cytogenetic analysis is necessary. Finally, investigating the effects of NaBu and DZNep in newly developed cell lines will further confirm the ability of the drugs to epigenetically reprogram class II cell lines to the class I.